Absorbable prophylactic composition for protection against ionizing or non-ionizing electromagnetic waves

ABSTRACT

An absorbable prophylactic composition for protecting against ionizing or nonionizing electromagnetic waves. The composition is derived from a crude extract of residual fractions of crushed grapes, and primarily from solid material comprising pips, stalks and skins of grapes and/or combinations thereof. The crude extract is obtained by first pulverizing the fraction or fractions from the grapes to form a powder, and then subjecting the powder to at least two extraction processes, one with acetone and the other with ethyl acetate. The extract is then ingested in doses between about 20 mg to 3 g for an adult male. Tests have demonstrated the efficacy of this composition against changes induced by ionizing radiation (x-rays) and nonionizing radiation (UV-C).

This application is a 371 of PCT/FR96/01577, filed Oct. 10, 1996.

The present invention concerns the use of residual fractions of crushedgrapes for preparing an absorbable prophylactic composition providingprotection against ionising or non-ionising electromagnetic waves.

The production of free radicals and, in particular, of oxygenated freeradicals is a normal physiological process at the cellular level notnormally having deleterious consequences since the human organism isprovided with protective systems: these protective systems areconstituted either by enzymatic systems that trap oxygenated freeradicals, such as SOD, or capable of decomposing secondary toxicproducts (H₂O₂) such as catalase, or by non-enzymatic systems such asvitamin E or glutathione.

It may happen, however, that the level of production of the oxygenatedfree radicals exceeds the capacities of the protective systems and theyreact with other molecules of the living cells, the consequences ofwhich may be profound disturbances of the membranal integrity, theinactivation of many enzymatic systems, and profound modifications tothe structure of the nucleic acids.

This hyper-production of oxygenated free radicals may result :

either from activation of endogenous mechanisms known to be generatorsof oxygenated free radicals, such as the energetic metabolism at thelevel of the respiratory chains or the activity of the macrophagesparticularly stimulated in the case of inflammation,

or from genesis induced by exogenous factors such as electromagneticwaves (ionising or non-ionising radiation) or hyper-oxygenation.

Among the mechanisms induced by excessive production of oxygenated freeradicals, scientists are in agreement in considering that the mostimportant is lipoperoxidation which corresponds to the degradation underthe effect of radical reactions of the polyunsaturated fatty acidspresent in the phospholipids constituting the membranes of living cells;as a consequence, these molecular species are the privileged target ofthe radical attacks.

It has been possible to determine experimentally that under the effectof oxygenated free radicals, the molecules of polyunsaturated fatty acidtend to oxidise and to generate numerous degradation products, amongwhich the most important are aldehydes, especially malondialdehyde. Theconsequence of this catabolism is a loss of the integrity and thestability of the polyunsaturated fatty acids, which manifests itself ina profound change in the cellular integrity.

It is generally accepted that these phenomena are involved inpathologies ass varied as rheumatoid polyarthritis, certain cancers,certain neuro-degenerative diseases (Alzheimers's, Parkinson's),cerebral and cardiac ischemias and, in general terms, ageing and celldeath.

Besides the above-mentioned classic protective systems (SOD, catalase,vitamin E, . . .), it has been known for several years that certainflavonoids of vegetable origin are excellent at trapping free radicals.It has been possible to ascertain that in this group the mostinteresting appear to be the procyanidols or proanthocyanidines whichcorrespond to hydrolysable tannins constituted by a glucidic molecule onwhich is esterified gallic acid or one of its derivatives and which arecapable of liberating cyanidol by hydrolysis in an acid medium.

According to their degree of increasing polymerisation, the procyanidolsare characterised by the following terms: monomers, dimers, trimers,tetramers, oligomers and condensates; among the monomers, the mostcommon are epigallocatechin and epigallocatechin gallate. It has in factbeen found that these compounds have a very high intrinsic efficacy andin particular are markedly more active than vitamin E.

Starting from this general knowledge, there has already been proposed,according to the publication JP-A-04/164 030, a prophylactic compositionfor protection against disorders linked to radioactivity—such as mayoccur for example in patients who are treated with radio-therapy, or inpermanent operators of apparatus emitting radioactive radiation—capableof being absorbed orally and the active constituents of which arepreviously purified epigallocatechin or epigallocatechin gallatemonomers. This Japanese publication indicates that this prophylacticcomposition may be constituted by a mixture of epigallocatechin andepigallocatechin gallate in a ratio of ⅓ obtained from leaves of greentea.

The preparation of this composition, however, has the drawback ofrequiring extraction and purification operations which are long andexpensive, and therefore particularly inconvenient.

According to the invention, the idea was conceived of remedying thesedrawbacks by the use, for preparing an absorbable prophylacticcomposition providing protection against ionising or non-ionisingradiation, not of a composition based on previously isolatedconstituents but of a crude extract of the residual fractions of crushedgrapes, that is to say, of a product available in large quantities andtherefore not difficult to obtain.

According to the invention, such a crude extract may contain pips and/orstalks and/or skins. It may also be obtained from residual press cakesfrom the manufacture of grapeseed oil.

It should be noted that in the course of wine-making operation thebunches of grapes are squashed, that is to say, partially crushed inorder to liberate the must which contains approximately 80% juice and20% solid matter essentially constituted by the pips, stalks and skins.This must is then transferred to a fermentation vat in which occur thedifferent chemical reactions of maceration and fermentation which permitthe transformation of the grape into wine. In the course of thesereactions, part of the constituents initially contained in the solidmatter diffuses into the juice. The residual solid matter is thenseparated from the juice then pressed so as to obtain a wine known as“press” wine, and a residue termed “marc”.

This residue may be distilled to produce brandy, or be used for animalfeed.

Another outlet for more or less dried wine-making marcs corresponds tothe manufacture of grapeseed oil by a pressing operation during thecourse of which the fatty part is separated from a press cake.

It should be noted that it has already been proposed to use by-productsor wine-making for the preparation, after purification, of a medicamentintended for the treatment of venous circulation problems: Endotelon®.

In spite of the various possibilities mentioned above, the major part ofthe wine-making marcs and of the press cakes from the manufacture ofgrapeseed oil is at present poorly exploited.

However, different earlier studies have made it possible to demonstratethat the grape pips present in these residues are particularly rich inprocyanidols and contain, in particular, large amounts ofepigallocatechin and epigallocatechin gallate which can be found inmonomer form but also in dimer, trimer and oligomer form.

According to the invention, it has been ascertained that theseprocyanidols are also at least partially present in the above-mentionedby-products of wine-making, besides other constituents such as catechin,or even gallic acid or caffeic acid.

It has consequently been proposed to use a crude extract of all or partsof the residual fraction of crushed grapes for preparing an absorbableprophylactic composition providing protection against ionising ornon-ionising electromagnetic waves.

It has been found that such a crude extract, surprisingly, has amarkedly greater efficacy than that which epigallocatechin andepigallocatechin gallate which are isolated, or even associated witheach other, have on the prevention of lipidic changes induced byradiation.

Taking into account the foregoing, the use according to the inventionhas the advantage of making it possible to derive value from theby-products of wine-making vines.

The invention also relates to an absorbable prophylactic compositionproviding protection against ionising or non-ionising electromagneticwaves.

According to the invention, this prophylactic composition ischaracterised in that it is constituted by a crude extract of all orparts of the residual fractions of crushed grapes containing essentiallycatechin, epicatechin and gallic acid, the catechin and epicatechinbeing able either to be associated with gallic acid or to be in anon-galloylated form, and these compounds being in the form of monomersor in the form of homogeneous or heterogeneous oligomers havingdifferent degrees of polymerisation, and in that it contains 10 to 30%of monomers, 10 to 30% of dimers, 10 to 30% of trimers and 30 to 60% oftetramers.

According to another characteristic of the invention, the compositioncontains traces of caffeic acid.

By way of a non-limiting example, such a crude extract may be obtainedby a process which is known per se, in which:

a preparation based on grape pips is pulverized,

the powder thus obtained is subjected to a first extraction withacetone,

the extract thus obtained is separated by filtration,

sodium chloride is added to it to saturation point,

the acetone is evaporated,

the extract is taken up again with distilled water,

the solution thus obtained is subjected to a second extraction withethyl acetate,

the organic phase is separated out and this is subjected tolyophilisation so as to obtain the crude extract.

It should be noted that it is of advantage to carry out thepulverisation of the base preparation in the presence of dry ice inorder to make it easier and to obtain greater homogenity.

It should also be noted that this base preparation is as a general rulerelatively rich in apolar compounds which it is advisable to eliminatein order to obtain a satisfaction composition at the end of the process.

To this end, it is of advantage to subject the solution to intermediateextraction with diethyl ether before carrying out the second extraction.

A base preparation of grape pips of Bordeaux origin was treated, by wayof example, by the above-mentioned process, using 70% acetone for thefirst extraction.

After lyophilisation, a composition was thus obtained containing 34% ofcatechin, 36% of epicatechin, 11.6% of gallic acid and 0.007% of caffeicacid.

It was also found that this composition contained 19% of monomers, 18%of dimers, 21% of trimers and 42% of tetramers.

Taking into account its good intestinal absorption and its rapiddiffusion in the organism, the daily dose “per os” of theabove-mentioned crude extract represents a regular nutritionalsupplement making it possible to combat the production of oxygenatedfree radicals induced by the action of radiation or oxydating stressexceeding the endogenous capacities for protection.

With regard to the recommendations for use, the quantity of extract tobe ingested may vary from case to case, but it is desirable as a generalrule to use daily doses of 20 mg to 3 g for an adult male.

The lower limit of this range applies by way of example to personsregularly exposed to weak radiation, while the upper limit is moreespecially suited to persons subjected to intense irradiation of shortduration.

Different tests have made it possible to demonstrate the efficacy of thecrude extract according to the invention against changes induced byionising radiation (X-rays) and non-ionising radiation (UV-C). Theeffficacy of this extract was first studied on pure polyunsaturatedfatty acids in aqueous solution after induction of the oxydating stressby UV-C rays (200 μW/cm²; 24 hrs). It was thus possible to obtain totalprotection of these molecules starting from 2 mg/l of extract in themedium.

A study was then made of the effect of this extract on the attacksinduced by UV-C rays on the membranal lipids of the brain and the liverof mice. There again, proof was provided of the total efficacy of thisextract starting from a concentration of 4 mg/l.

In a third step, a study was made of the effect of UV-C radiation andX-rays on human fibroblastic cells in culture and also the protectionprovided by the crude extract according to the invention. Observation ofthe cellular lysis after UV-C irradiation (200 μW/cm²; 1 hr) showed thatthe protection provided by this extract for the cells in culture wasdose-dependent and that it is greater than 50% for a concentration ofextract of 40 mg/l.

For X-rays, the cellular viability was taken into consideration. Thisindex was evaluated by cellular enumeration, but also by observing thepower of proliferation of the cells placed in culture again afterirradiation with X-rays (30 Grays at the rate of 2 Gy/min). This made itpossible to ascertain that, starting from 20 mg/l of crude extract inthe medium, the number of dead cells is significantly reduced. Inaddition, the proliferation of the cells which is blocked in the cellsirradiated in the absence of extract is to a great extent restored inthe cells irradiated in the presence of this extract.

Finally, a study was made of the effect of the extract according to theinvention on the haematopoietic system after total irradiation of malerats with X-rays (5 Gy in 3 sessions). Previously, a series of rats wereforce-fed with 60 mg/day of extract. Six days after the lastirradiation, the animals were anaesthetized and the blood was sampled atthe carotid. Blood analysis was carried out on each of the samples.

Before irradiation, no toxic effect was observed in the animals havingingested the preparation according to the invention.

The results of this experiment showed that there was a reduction in allthe blood constituents and parameters (red blood cells, platelets,haematocrits, haemoglobin, polyneutrophiles, lymphocytes and monocytes)in the rats irradiated without pre-treatment, and that there was amarked improvement in the animals previously force-fed with the crudeextract according to the invention.

By way of example, a platelet count was carried out on control animals,irradiated animals and irradiated animals previously treated with theextract according to the invention. The following results were thusobtained:

control animals  327.5 ± 103.5 G/l irradiated animals 152.66 ± 49.93 G/lanimals treated then irradiated 277.66 ± 78.54 G/l

These results clearly show the efficacy in vivo of the extract accordingto the invention.

In order to highlight the superiority of the crude extract according tothe invention compared with isolated epigallocatechin andepigallocatechin gallate, according to the publication JP-A-04/164 030,a preparation of microsomes of baboon liver was irradiated with UV-C(200 μW/cm²) for 15 hours in the presence of epigallocatechin (EGC), ofepigallocatechin gallate (EGCG) or of the crude extract according to theinvention (I).

The additions were made in such a way as always to have the samequantity of epicatechin, that is to say, 1.45 mg/l.

The residual polyunsaturated fatty acids (AGPI) (20:4 n−6 and 22:6 n−3)were then quantified and also malondialdehyde (MDA) which is a productof their degradation.

This test allowed the following results to be obtained:

AGPI Concentration MDA (μM) 20:4 n − 6 (μg/ml) 22:6 n − 3 (μg/ml)Non-irradiated 0 137.85 ± 2.65 42.43 ± 14.0 controls Samples irra- 34.17± 4.07 081.73 ± 7.65 15.40 ± 1.57 diated without additive Samples irra-22.66 ± 1.75 136.29 ± 0.08 45.25 ± 9.61 diated + I Samples irra- 32.02 ±2.60 094.53 ± 9.11 24.05 ± 2.36 diated + EGC Samples irra- 32.25 ± 0.76098.59 ± 21   31.40 ± 1.48 diated + EGCG

These results prove that irradiation of the microsomes results indegradation of the polyunsaturated fatty acids and an increase in theproduction of malondialdehyde.

The extract according to the invention appears to provide significantprotection for the microsomes against these changes, which is not thecase with isolated epigallocatechin and epigallocatechin gallate.

The same test was then repeated, irradiating a preparation of microsomesof baboon liver at a lower proteic concentration in the presence of thecrude extract of grape pips according to the invention and of a mixtureconsisting of ¼ epigallocatechin and ¾ epigallocatechin gallate.

The additions were once again carried out in such a way as to have 1.45mg/l of epicatechin in all cases.

The analyses carried out, similar to the above-mentioned test, allowedthe following results to be obtained:

AGPI 20:4 n − 6 Concentration MDA (μM) (μg/ml) 22:6 n − 3 (μg/mlNon-irradiated 0.378 ± 0.05 18.96 ± 0.86 8.21 ± 1.85 controls Samplesirra- 14.93 ± 0.35  6.66 ± 0.11 undetectable diated without additiveSamples irra-  5.91 ± 1.76 16.59 ± 0.01 7.51 ± 0.47 diated + I Samplesirra- 14.186 ± 0.37  09.59 ± 1.15 2.95 ± 0.99 diated + EGC/ EGCG mixture

This text again made it possible to prove that the extract according tothe invention allows protection of the microsomes against the changesinduced by UV-C rays, while the epigallocatechin/epigallocatechingallate mixture appears totally ineffective at the doses used.

What is claimed is:
 1. A method of making absorbable prophylacticcomposition from solid material comprising a crude extract derived fromthe pips, stalks, and/or skins and/or combinations thereof from crushedgrapes, and/or residual press cakes from the manufacture of grapeseedoil and/or combinations thereof for protection against ionizing ornonionizing radiation, said method comprising: a) pulverizing said solidmaterial to form powdered material; b) subjecting the powdered materialto a first extraction with acetone to provide an extract; c) separatingthe extract; d) adding sodium chloride to the extract; e) evaporatingthe acetone from the extract; f) mixing the extract with water to form asolution; g) subjecting the solution to a second extraction with ethylacetate to obtain an organic phase; h) separating the organic phase andsubjecting the organic phase to lyophilisation to provide saidprophylactic composition.
 2. The method of claim 1, wherein the solidmaterial extracted from the grapes comprises pips, stalks, skins, orcombinations thereof.
 3. The method as recited in claim 1, wherein thematerial extracted from grapes comprises pips.
 4. The method as recitedin claim 1, wherein the material extracted from grapes is subjected toextraction with diethyl ether before the second extraction.
 5. Themethod as recited in claim 4, wherein subjecting the material toextraction with diethyl ether is accomplished prior to subjecting thesolution to the extraction with ethyl acetate.
 6. The method as recitedin claim 1, further comprising pulverizing the solid material extractedfrom grapes in the presence of a freezing medium.
 7. The method asrecited in claim 6, wherein said freezing medium comprises dry ice. 8.An absorbable prophylactic composition made in accordance with a methodrecited in claim
 1. 9. The composition as recited in claim 8, whereinsaid composition is derived from solid material extracted from grapeswhich contains catechin, epicatechin and gallic acid, the catechin andepicatechin being able to be intermixed with the gallic acid or to be ina nongalloylated form.
 10. The composition as recited in claim 9,wherein the catechin, epicatechin and gallic acid are in the form ofmonomers in the form homogeneous or heterogeneous oligomers havingdifferent degrees of polymerization.
 11. The composition as recited inclaim 8, wherein the solid material extracted from grapes contains 10%to 30% monomers, 10 to 30% dimers, 10 to 30% of trimers, and 30 to 60%tetramers.
 12. The composition as recited in claim 11, wherein the solidmaterial extracted from grapes contains traces of caffeic acid.
 13. Aprophylactic composition for protection against ionizing or nonionizingradiation, which comprises a crude extract derived from the pips,stalks, and/or skins and/or combinations thereof from crushed grapes,and/or residual press cakes from the manufacture of grapeseed oil and/orcombinations thereof, comprising catechin, epicatechin and gallic acid,the catechin and epicatechin being able either to be intermixed withgallic acid or to be in nongalloviated form.
 14. The composition asrecited in claim 13 wherein the catechin and epicatechin and gallic acidis in the form of monomers or in the form of homogeneous orheterogeneous oligomers.
 15. The composition as recited in claim 14,wherein the composition contains 10 to 30% of monomers, 10 to 30%dimers, 10 to 30% of trimers, and 30 to 60% tetramers.
 16. A method ofproviding protection against ionizing or nonionizing radiation byingesting a therapeutic dose of a composition derived from pips, stalks,and/or skins from crushed grapes and/or combinations thereof, and/orresidual press cakes from the manufacture of grapeseed oil wherein saidcomposition comprises a solid material extracted from grapes whichcontains catechin, epicatechin and gallic acid, the catechin andepicatechin being able to be intermixed with the gallic acid or to be ina nongalloylated form.
 17. The method as recited in claim 16, whereinthe catechin, epicatechin and gallic acid are in the form of monomers inthe form of homogeneous or heterogeneous oligomers.
 18. The method asrecited in claim 17, wherein said composition contains 10% to 30%monomers, 10 to 30% dimers, 10 to 30% of trimers, and 30 to 60%tetramers.
 19. The method as recited in claim 18, wherein saidcomposition comprises the solid material extracted from grapes containstraces of caffeic acid.